Nucleic acid amplification system, biological sample RNA in-situ imaging method and application

Abstract

The invention provides a nucleic acid amplification system, a biological sample RNA in-situ imaging method and application, the nucleic acid amplification system is realized based on the combination of CRISPR and isothermal amplification technology, the nucleic acid amplification system comprises: a CRISPR improvement module, the CRISPR improvement module comprises dCas13d, split-crL, split-crR, circle DNA and a Cas13 reaction buffer solution; a rolling circle amplification module, wherein the rolling circle amplification module comprises a phi29 DNA polymerase and a phi29 reaction buffer solution; a fluorescent probe module; nucleic acid to be amplified firstly enters the CRISPR improvement module, then enters the rolling circle amplification module and finally enters the fluorescent probe, and the nucleic acid detection signal in the cell can be rapidly amplified and RNA imaging can be carried out in a high-sensitivity and high-specificity mode.

Description

Technical Field

[0001] The present invention relates to the technical field of bioimaging technology, and in particular, to a nucleic acid amplification system, a method for in-situ imaging of RNA in biological samples, and applications thereof.

Background Art

[0002] The fluorescence in-situ imaging technology of RNA is a powerful molecular biology tool for directly observing and localizing the presence and distribution of specific RNA molecules in cell or tissue samples. This technology allows researchers to precisely observe the spatial location of RNA in cells by using complementary labeled probes to specifically bind to the target RNA sequence and then visualizing it through a fluorescence microscope or other imaging systems.

[0003] However, despite the significant advantages of this technology in terms of precision and intuitiveness, there are still several limitations in its practical applications: 1. Although traditional methods such as smFISH or RNAscope can perform imaging at the single-molecule level, many fluorescent probes need to be designed to achieve this, so the target RNA to be detected requires a relatively long targeting region; 2. Other methods, such as Basescope or the method of rolling circle amplification through DNA sequence binding ligase, although they can target shorter targets, are limited by the ligation efficiency of ligase and the nucleic acid hybridization efficiency, resulting in a decrease in detection sensitivity; 3. For detecting the effects of some RNAs with special structures (such as circular RNAs), existing RNA imaging methods are not ideal.

[0004] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology is a revolutionary gene editing tool. Currently, this technology has been widely applied in multiple fields such as gene editing, biological detection, gene therapy, molecular diagnosis, and nucleic acid imaging. Among them, Cas13 protein can specifically target RNA rather than DNA, making it an ideal tool for studying gene expression regulation and RNA-related diseases.

[0005] Isothermal amplification is a technology for nucleic acid amplification under constant temperature conditions without the temperature cycling in traditional PCR. Among many isothermal amplification methods, rolling circle amplification (RCA) has attracted much attention due to its unique advantages. RCA uses a specific DNA polymerase (such as phi29 DNA polymerase) to efficiently and continuously replicate circular templates, showing characteristics such as high fidelity, high amplification efficiency, and simplicity of operation. In addition, RCA can generate ultra-long and repetitive DNA sequence products, having broad application potential.

[0006] Therefore, it is necessary to study a nucleic acid amplification system, a method and application for in-situ imaging of RNA in biological samples to address the deficiencies of the prior art and to solve or mitigate one or more of the above problems.

Summary of the Invention

[0007] In view of this, to solve the problem that the in-situ hybridization imaging technology currently used for fixing intracellular RNA cannot simultaneously achieve high specificity, high sensitivity, and imaging of complex RNA structures, the present invention provides a nucleic acid amplification system, a method and application for in-situ imaging of RNA in biological samples, which can rapidly, highly sensitively, and highly specifically amplify the nucleic acid detection signal in cells and perform RNA imaging.

[0008] On the one hand, the present invention provides a nucleic acid amplification system, which is based on the combination of CRISPR and isothermal amplification technologies. The nucleic acid amplification system includes:

[0009] A CRISPR improvement module for further improving crRNA. The CRISPR improvement module includes dCas13d, split-crL, split-crR, circle DNA, and Cas13 reaction buffer;

[0010] A rolling circle amplification module for performing an amplification reaction. The rolling circle amplification module includes phi29 DNA polymerase and phi29 reaction buffer;

[0011] A fluorescent probe module for labeling the amplified nucleic acid;

[0012] The nucleic acid to be amplified first enters the CRISPR improvement module, then enters the rolling circle amplification module, and finally enters the fluorescent probe.

[0013] In the aspect and any possible implementation manner as described above, a further implementation manner is provided. The CRISPR improvement module activates the targeting activity of the dCas13d protein by performing a ring-opening treatment on the conserved region loop part of crRNA and extending two hybridization sequences for recombination.

[0014] In the aspect and any possible implementation manner as described above, a further implementation manner is provided. The circleDNA serves as a template for rolling circle amplification RCA and binds to the improved crRNA to achieve signal amplification.

[0015] In the aspect and any possible implementation manner as described above, a further implementation manner is provided. The phi29 DNA polymerase is used to efficiently amplify circle DNA to generate an extremely long repetitive DNA sequence.

[0016] For the aspects and any possible implementation manners described above, a further implementation manner is provided, where the dCas13d protein is used to target RNA.

[0017] For the aspects and any possible implementation manners described above, a further implementation manner is provided, where the fluorescent probe module includes, but is not limited to, a Cy3-labeled fluorescent probe.

[0018] For the aspects and any possible implementation manners described above, a further implementation manner is provided for a method of in-situ imaging of RNA in a biological sample. In-situ imaging is achieved through the nucleic acid amplification system described above. The method of in-situ imaging includes the following steps:

[0019] S1: Improve the CRISPR system to activate the targeting activity of the dCas13d protein;

[0020] S2: Rolling circle amplification to generate an ultra-long repetitive DNA sequence;

[0021] S3: Fluorescent probe hybridization, where the Cy3-labeled fluorescent probe hybridizes with the RCA amplification product to achieve fluorescence imaging.

[0022] For the aspects and any possible implementation manners described above, a further implementation manner is provided, where S1 specifically includes:

[0023] S11: split-crRNA design, where the targeting activity of the dCas13d protein is activated by opening the loop part of the conserved region of crRNA and extending two hybridization sequences for recombination;

[0024] S12: circle DNA binding, where the improved crRNA design includes a sequence for binding to circle DNA to achieve RCA amplification.

[0025] For the aspects and any possible implementation manners described above, a further implementation manner is provided, where S2 specifically includes efficiently amplifying circle DNA by phi29 DNA polymerase at a constant temperature to generate an ultra-long repetitive DNA sequence.

[0026] For the aspects and any possible implementation manners described above, a further implementation manner is provided for the application of a method of in-situ imaging of RNA in a biological sample. RNA detection is performed through the method of in-situ imaging described in any one of the above.

[0027] Compared with the prior art, the present invention can achieve the following technical effects:

[0028] 1. Through the improved CRISPR system and rolling circle amplification (RCA) technology, the present invention realizes high-sensitivity and high-specificity in-situ imaging of intracellular RNA, which is applicable to the detection of complex RNA structures.

[0029] 2. It solves the limitations of multiple groups of targeting probes and complex sequence design required by traditional fluorescence in situ hybridization. Only one targeting probe is needed in the present invention to complete imaging.

[0030] 3. It solves the limitations of the binding efficiency of padlock probes and the ligation efficiency of ligases when using traditional rolling circle amplification (RCA) imaging. High-efficiency recognition can be achieved by the CRISPR system in the present invention.

[0031] 4. The operation of the present invention is simple and the reaction is fast, solving the drawback of the long reaction time (>1 day) of traditional fluorescence in situ hybridization.

[0032] Of course, it is not necessary for any product implementing the present invention to achieve all the above-mentioned technical effects simultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0034] Figure 1 It is a schematic diagram of the imaging method for RNA in fixed cells provided by an embodiment of the present invention;

[0035] Figure 2 It is a confocal image of the detection of EGFP mRNA in HEK293T-EGFP (+EGFP) and HEK293T (-EGFP) using the present invention provided by an embodiment of the present invention; The first column is the merged fluorescence image of Cy3 and DAPI fluorescence, with excitation wavelengths of 552 nm (Cy3) and 405 nm (DAPI), the second column is EGFP fluorescence, with an excitation wavelength of 488 nm, and the scale bar is 1 μm;

[0036] Figure 3 It is a confocal image of the detection of TK1 mRNA in MCF-7 (the first row) and MCF-10A (the second row) using the present invention provided by an embodiment of the present invention; The first column is Cy3 fluorescence, with an excitation wavelength of 552 nm, the second column is DAPI fluorescence, with an excitation wavelength of 405 nm, and the last column is the merged fluorescence image, and the scale bar is 20 μm.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] In order to better understand the technical solutions of the present invention, the embodiments of the present invention will be described in detail below in conjunction with the drawings.

[0038] It should be clear that the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0039] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "the" and "said" used in the embodiments of the present invention and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0040] The present invention provides a nucleic acid amplification system, which is realized based on the combination of CRISPR and isothermal amplification technologies. The nucleic acid amplification system includes:

[0041] A CRISPR improvement module for further improving crRNA. The CRISPR improvement module includes dCas13d, split-crL, split-crR, circle DNA and Cas13 reaction buffer;

[0042] A rolling circle amplification module for performing an amplification reaction. The rolling circle amplification module includes phi29 DNA polymerase and phi29 reaction buffer;

[0043] A fluorescent probe module for labeling the amplified nucleic acid;

[0044] The nucleic acid to be amplified first enters the CRISPR improvement module, then enters the rolling circle amplification module, and finally enters the fluorescent probe.

[0045] The CRISPR improvement module activates the targeting activity of the dCas13d protein by performing an open-loop treatment on the conserved region neck loop part of crRNA, extending two hybridization sequences and recombining them.

[0046] The circle DNA serves as a template for rolling circle amplification RCA, binds to the improved crRNA, and realizes signal amplification.

[0047] The phi29 DNA polymerase is used to efficiently amplify circle DNA to generate ultra-long repetitive DNA sequences.

[0048] The dCas13d protein is used to target RNA.

[0049] The fluorescent probe module includes but is not limited to a Cy3-labeled fluorescent probe.

[0050] The present invention also provides a method for in-situ imaging of RNA in biological samples, which realizes in-situ imaging through the nucleic acid amplification system described above. The in-situ imaging method includes the following steps:

[0051] S1: Improve the CRISPR system to activate the targeting activity of the dCas13d protein;

[0052] S2: Rolling circle amplification to generate ultra-long repetitive DNA sequences;

[0053] S3: Fluorescent probe hybridization. The Cy3-labeled fluorescent probe hybridizes with the RCA amplification product to achieve fluorescence imaging.

[0054] The specific steps of S1 include:

[0055] S11: split-crRNA design. By opening the loop of the conserved region of the crRNA and extending two hybridization sequences for recombination, the targeting activity of the dCas13d protein is activated;

[0056] S12: circle DNA binding. The improved crRNA design includes sequences for binding to circle DNA to achieve RCA amplification.

[0057] The specific steps of S2 include efficiently amplifying circle DNA by phi29 DNA polymerase at a constant temperature to generate ultra-long repetitive DNA sequences.

[0058] The present invention also provides an application of a method for in-situ imaging of RNA in biological samples. RNA detection is performed by any of the in-situ imaging methods described above.

[0059] The imaging principle of the present invention is as follows:

[0060] The present invention improves the original dCas13d system. By opening the loop of the conserved region of the crRNA and extending two hybridization sequences for recombination, the targeting activity of the dCas13d protein is activated, and under its guidance, the crRNA is directed to the target RNA. In addition, the improved crRNA design includes sequences for binding to circle DNA, which enables an amplification reaction to be carried out by rolling circle amplification (RCA) under the action of phi29 DNA polymerase. Finally, the fluorescent probe is hybridized to the product after RCA amplification, and imaging is performed by a confocal microscope.

[0061] The imaging amplification system of the present invention comprises the following raw materials: dCas13d, split-crL, split-crR, circleDNA, Cas13 reaction buffer, phi29 DNA polymerase, phi29 reaction buffer, dNTP mix, fluorescent probe, hybridization buffer. The RNA and DNA sequences used are shown in Table 1.

[0062] Table 1 Oligonucleotide sequences

[0063]

[0064] Note: (1) The direction of the DNA sequence is from the 5'-end to the 3'-end. (2) Cy3 represents fluorescent group modification.

[0065] The oligonucleotide sequences in Table 1 were all provided by Sangon Biotech (Shanghai) Co., Ltd. through HPLC purification.

[0066] The method for detecting RNA using the above imaging principle includes the following steps:

[0067] (1) Fix the cells with 4% paraformaldehyde fixative at room temperature.

[0068] (2) After fixation, permeabilize the cells with PBS containing Triton X-100, and then wash the cells with PBS.

[0069] (3) Add the dCas13 reaction buffer (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, pH 7.9) containing dEsCas13d, split-crL, split-crR, and circle DNA to the cells, incubate at 37°C for 30 min, and then wash the cells once with buffer A.

[0070] (4) Add the phi29 reaction buffer containing phi29 DNA polymerase and dNTP mix to the cells, incubate at 37°C for 30 min, and then wash the cells once with buffer A.

[0071] (5) Add the hybridization buffer containing the fluorescent probe to the cells, incubate at 37°C for 30 min, and then wash the cells once with buffer A and once with buffer B.

[0072] (6) Stain the cell nuclei with DAPI and then perform confocal imaging.

[0073] Example 1:

[0074] In-situ imaging of EGFP mRNA in HEK293T cells (HEK293T-EGFP) stably transfected with the EGFP gene was performed as follows:

[0075] (1) Wash the cells cultured in a 12-well plate 3 times with 1 ml of 1xPBS.

[0076] (2) Fix the HEK293T-EGFP and HEK293T cells with 4% paraformaldehyde fixative at room temperature.

[0077] (3) After fixation, the cells were permeabilized with 1×PBS containing 0.5% Triton X-100 at room temperature for 5 min, and then the cells were washed 3 times with 1×PBS;

[0078] (4) The dCas13 reaction buffer containing 2 μM dEsCas13d, 1 μM split-crL, 1 μM split-crR-EGFP, and 1 μM circleDNA was added to the cells and incubated at 37 °C for 30 min, and then the cells were washed once with bufferA;

[0079] (5) The phi29 reaction buffer containing phi29 DNA polymerase and dNTP mix (1 mM each) was added to the cells and incubated at 37 °C for 30 min, and then the cells were washed once with bufferA;

[0080] (6) The hybridization buffer containing the fluorescent probe was added to the cells and incubated at 37 °C for 30 min, and then the cells were washed once with bufferA and then once with bufferB;

[0081] (7) The nuclei were stained with DAPI and then confocal imaging was performed. The results are as Figure 2 shown. It can be seen from the figure that fluorescent signals only appeared in the cells containing EGFP, indicating that the present invention has a good imaging effect on stably transfected mRNA targets with high expression levels, and also proves the feasibility of the invention.

[0082] Example 2:

[0083] In situ imaging of TK1 mRNA in MCF-7 cells was performed as follows:

[0084] (1) The cells cultured in a 12-well plate were washed 3 times with 1 ml of 1xPBS;

[0085] (2) MCF-7 and MCF-10A cells were fixed with 4% paraformaldehyde fixative at room temperature;

[0086] (3) After fixation, the cells were permeabilized with 1×PBS containing 0.5% Triton X-100 at room temperature for 5 min, and then the cells were washed 3 times with 1×PBS;

[0087] (4) Add the dCas13 reaction buffer containing 2 μM dEsCas13d, 1 μM split-crL, 1 μM split-crR-TK1, and 1 μM circular DNA to the cells and incubate at 37 °C for 30 min. Then wash the cells once with buffer A;

[0088] (5) Add the phi29 reaction buffer containing phi29 DNA polymerase and dNTP mix (1 mM each) to the cells and incubate at 37 °C for 30 min. Then wash the cells once with buffer A;

[0089] (6) Add the hybridization buffer containing the fluorescent probe to the cells and incubate at 37 °C for 30 min. Then wash the cells once with buffer A and then once with buffer B;

[0090] (7) Stain the cell nuclei with DAPI and then perform confocal imaging. The results are as Figure 3 shown. It can be seen from the figure that obvious fluorescence signals were only generated in MCF7 cells, indicating that the present invention also has a good imaging effect on mRNA targets with low expression levels such as TK1.

[0091] The above has introduced in detail a nucleic acid amplification system, a method for in-situ imaging of biological sample RNA and its application provided by the embodiments of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application; at the same time, for those of ordinary skill in the art, according to the idea of the present application, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present application.

[0092] As used in the specification and claims, certain terms are used to refer to specific components. Those skilled in the art should understand that hardware manufacturers may use different terms to refer to the same component. The specification and claims do not use the difference in names as a way to distinguish components, but use the difference in functions of components as the criterion for distinction. As mentioned throughout the specification and claims, "comprising" and "including" are open-ended terms, so they should be interpreted as "comprising / including but not limited to". "Substantially" means within an acceptable error range. Those skilled in the art can solve the technical problem within a certain error range and basically achieve the technical effect. The subsequent description in the specification is the preferred implementation manner for implementing the present application, but the description is for the purpose of explaining the general principles of the present application and is not used to limit the scope of the present application. The protection scope of the present application shall be determined by the scope defined by the appended claims.

[0093] It should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a commodity or system comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or elements inherent to such commodity or system. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the commodity or system comprising said element.

[0094] It should be understood that the term "and / or" used herein is merely a description of the relationship between associated objects, indicating that three relationships may exist. For example, A and / or B may represent: the sole existence of A, the simultaneous existence of A and B, and the sole existence of B. Additionally, the character " / " herein generally represents an "or" relationship between the associated objects before and after.

[0095] The above description illustrates and describes several preferred embodiments of the present application. However, as previously mentioned, it should be understood that the present application is not limited to the forms disclosed herein, should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be changed within the scope of the application concept described herein through the above teachings or the techniques or knowledge in the relevant field. Any changes and variations made by those skilled in the art without departing from the spirit and scope of the present application shall fall within the protection scope of the appended claims of the present application.

Claims

1. A nucleic acid amplification system based on the combination of CRISPR and isothermal amplification technology, characterized in that: The nucleic acid amplification system comprises: A CRISPR improvement module, used to further improve crRNA, wherein the CRISPR improvement module includes dCas13d, split-crL, split-crR, circle DNA and Cas13 reaction buffer; A rolling circle amplification module, used for performing an amplification reaction, wherein the rolling circle amplification module comprises phi29 DNA polymerase and phi29 reaction buffer; A fluorescent probe module, used to label the amplified nucleic acid; The nucleic acid to be amplified first enters the CRISPR improvement module, then the rolling circle amplification module, and finally the fluorescent probe.

2. The nucleic acid amplification system according to claim 1, characterized in that The CRISPR improvement module opens the conserved region neck loop of crRNA and extends two hybridization sequences for recombination, thereby activating the targeting activity of dCas13d protein.

3. The nucleic acid amplification system according to claim 1, characterized in that: The circle DNA is used as a template for rolling circle amplification (RCA) and combines with the improved crRNA to achieve signal amplification.

4. The nucleic acid amplification system according to claim 1, characterized in that: The phi29 DNA polymerase is used for efficiently amplifying circle DNA and generating ultra-long repetitive DNA sequences.

5. The nucleic acid amplification system according to claim 1, characterized in that: The dCas13d protein is used to target RNA.

6. The nucleic acid amplification system according to claim 5, characterized in that: The fluorescent probe module includes but is not limited to a fluorescent probe labeled with Cy3.

7. A method for in situ imaging of RNA in a biological sample, which is achieved by using the nucleic acid amplification system according to any one of claims 1 to 6, characterized in that: The in situ imaging method comprises the following steps: S1: Improve the CRISPR system to activate the targeting activity of dCas13d protein; S2: rolling circle amplification to generate ultra-long repetitive DNA sequences; S3: Fluorescent probe hybridization: Cy3-labeled fluorescent probe hybridizes with the RCA amplification product to achieve fluorescent imaging.

8. The in-situ imaging method according to claim 7, characterized in that: The S1 specifically includes: S11: Split-crRNA design, by opening the conserved region of crRNA neck loop and extending two hybrid sequences for recombination, activating the targeting activity of dCas13d protein; S12: circle DNA binding, the improved crRNA design includes sequences for binding to circle DNA to achieve RCA amplification.

9. The in-situ imaging method according to claim 7, characterized in that: The S2 specifically includes efficiently amplifying circle DNA at a constant temperature by phi29 DNA polymerase to generate ultra-long repetitive DNA sequences.

10. An application of a biological sample RNA in situ imaging method, characterized in that: RNA detection is performed by the in situ imaging method as described in any one of claims 7 to 9.

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